Pertussis is a serious and potentially life-threatening respiratory disease caused by the bacterium Bordetella pertussis. Worldwide, 18.4 million people become ill with pertussis each year, and 254,000 of these patients die. Despite high vaccine coverage in the United States, there were still over 48,000 pertussis cases reported in the U.S. in 2012. Rapid diagnosis of pertussis is critical because treatment and outbreak containment are only effective if initiated early in the disease. Currently, there are no assays that can rapidly and accurately detect early-stage pertussis. The goal is to develop a rapid, point-of-care (POC) immunoassay to detect B. pertussis during early disease. The target population will be infants who present with the non-specific, respiratory tract infection symptoms of early pertussis (prior to onset of paroxysmal cough). The approach will be detection of B. pertussis antigens from nasopharyngeal swabs by lateral flow immunoassay (LFI). The product will be an assay relevant to i) U.S. pediatrician offices, and ii) international clinics in low-resource settings: a sensitive and specific test that delivers results in 5-10min at very low cost without specialized equipment or user expertise. In preliminary studies, we used a bioinformatics analysis to identify five high priority B. pertussis protein antigens for further stdy. Specific Aim 1 will develop epitope-specific polyclonal antibodies (pAbs) for detection of B. pertussis antigens. pAbs will be screened to select those that i) react with B. pertussis proteins, ii) react with B. pertussis but not other bacteria found in the nasopharynx, iii) can function in pairs to bind the antigen in the sandwich format necessary for LFI construction, and iv) detect antigen in patient nasopharyngeal samples. Only pAbs that meet all the criteria will be advanced to LFI prototype development in Specific Aim 2. Specific Aim 2 will determine the limit of detection of B. pertussis by LFI by optimizing the physical and biological components of the LFI prototypes. Based on previously determined B. pertussis loads in infant NP swabs of 106 bacteria per swab, the benchmark for sensitivity is detection of 105 CFU per swab. Together, the experiments of Phase I will determine which B. pertussis antigen epitopes can produce pAb pairs that yield LFI prototypes with the required sensitivity. A Phase II study would develop monoclonal antibodies (mAbs) against the validated epitopes, incorporate them into an LFI, and perform detailed pre-clinical studies. Successful navigation of a full clinical trial in Phase III would ultimately yield a POC immunoassay that could dramatically increase early pertussis diagnosis, limit pertussis transmission during community outbreaks, and save patient lives.